Spectroscopic analyses on interaction of o-Vanillin-d-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-L-Levodopa Schiff Bases with bovine serum albumin (BSA)

Article abstract of DOI:10.1016/j.saa.2010.12.077

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-d- Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were

Full text of DOI:10.1016/j.saa.2010.12.077

Spectrochimica Acta Part A 78 (2011) 1278–1286
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic analyses on interaction of o-Vanillin-d-Phenylalanine,
o-Vanillin-l-Tyrosine and o-Vanillin-l-Levodopa Schiff Bases with bovine serum
albumin (BSA)
Jingqun Gaoa, Yuwei Guoa, Jun Wanga,∗, Zhiqiu Wanga, Xudong Jina, Chunping Chengb, Ying Lia, Kai Lia
a Department of Chemistry, Liaoning University, Shenyang 110036, PR China
b Department of Chemistry, Baotou Normal College, Baotou 014030, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-
Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL)) with alanine constituent were synthesized by direct
reflux method in ethanol solution, and then were used to study the interaction to bovine serum albu-
min (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the
bimolecular quenching constant (Kq), apparent quenching constant (Ksv), effective binding constant (KA)
and corresponding dissociation constant (KD) as well as binding site number (n) were obtained. In addi-
tion, the binding distance (r) was also calculated according to Foster’s non-radioactive energy transfer
theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the bind-
ing array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates
that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr)
residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.
© 2010 Elsevier B.V. All rights reserved.
Received 13 October 2010
Received in revised form
14 December 2010
Accepted 25 December 2010
Keywords:
Interaction
Bovine serum albumin (BSA)
o-Vanillin Schiff Bases
Fluorescence spectroscopy
1. Introduction
play obvious bioactivities in experimental stage, they are usually
unsatisfactory in animal studies or clinical stage due to a lack of
Aromatic Schiff Bases are a large class of organic compounds
being from condensation reaction of aromatic aldehyde and amine
[1–3]. The –HC N– group and special plane structure make Aro-
matic Schiff Bases have the function of antibacterial, antitumour
and antivirotic activities [4–6]. Therefore, more and more Aro-
matic Schiff Bases have already been researched purposively and
developed as potential antibacterial, antitumour and antivirotic
drugs in recent years [7–14]. 2-Hydroxy-3-methoxybenzaldehyde
(o-Vanillin) is a kind of important chemical-industrial raw mate-
rial [15–17]. Because of many biological activities, such as analgesic,
anti-inflammatory, antibacterial, sterilizing and antiviral activities
[18–23], o-Vanillin is a useful medicine chemical engineering and
organic synthesis midbody [24–27]. In addition, it also can be used
as efficient herbicide, pesticide and bactericide [28–31]. Hence, o-
Vanillin is a optimal candidate for synthesizing various Aromatic
Schiff Bases with important bioactivities.
targeting. It has been well known that the amino acids are main
component element of various proteins. They generally play an
important physiological role in life process [32–34]. Because of
special three-dimensional geometric configuration, all amino acids
also exhibit the perfect identification and selection abilities to
biological tissue [35–37]. In order to further investigate the struc-
ture, composition and chemical and biologic activities of Aromatic
Schiff Bases, it is necessary to adopt some amino acids as amine
part for synthesizing new o-Vanillin Aromatic Schiff Bases. Hence,
in this work, three amino acids, such as 3-phenyl-d-alanine (d-
Phenylalanine), 3-(4-hydroxyphenyl)-l-alanine (l-Tyrosine) and
3-(3,4-dihydroxyphenyl)-l-alanine (l-Levodopa), were used to
react with o-Vanillin and three new o-Vanillin Schiff Bases (o-VSB:
o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine (o-VLT)
and o-Vanillin-l-Levodopa (o-VLL)) were synthesized. They would
display both natural biological activities and identification and
selection abilities to biological molecules.
Apart from deoxyribonucleic acid (DNA), many functional pro-
teins could also been considered as a target or carrier in various
drug designs. It has been well known that the serum albumins are
the most abundant proteins in the plasma [38–40]. It also has many
physiological functions. In the present work, the bovine serum
albumin (BSA) was chosen as a target protein molecule because of
its low cost, ready availability and unusual ligand-binding prop-
However, for a long time many researches were only focused
on the bioactivities of aromatic aldehyde and –HC N– group in
Aromatic Schiff Bases, and did not pay attention to their targeting
to biological tissue [8]. Although the Aromatic Schiff Bases dis-
∗
Corresponding author. Tel.: +86 024 6220783x7859; fax: +86 024 62202053.
E-mail address: wangjun890@126.com (J. Wang).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.12.077

J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
1279
Fig. 1. Molecular structure of o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL).
erties [41–44]. And what’s more, the whole structures of BSA
molecule are similar to human serum albumin (HSA) molecule in
76%, so the results of all the studies are considered to be consis-
tent with the facts that used HSA as the study model [45]. In this
study, the quenching of intrinsic fluorescence of proteins was used
to research the interaction between BSA molecules and three syn-
thesized o-VSB (o-VDP, o-VLT and o-VLL). Based on the obtained
parameters, the binding degrees were compared. Meanwhile, the
influences of substituent group on the interaction were also dis-
cussed. Besides, the binding sites of o-VSB (o-VDP, o-VLT and o-VLL)
to BSA molecules were also explored by synchronous fluorescence
spectroscopy. The molecular structures of three synthesized o-VSB
are shown in Fig. 1.
(Cary 50, Varian Company, USA). The compositions of o-Vanillin
Schiff Bases (o-VSB) were determined by using elementalanal-
yser (Perkin-Elmer 2400, PerkinElmer Company, USA). And their
structures were analyzed by Fourier transform infrared spec-
trophotometer (Spectrum 100, Perkin-Elmer Company, USA). The
solution pH value was measured with pH meter (PHS-3C, Shanghai
Leici Instrument Company, Ltd, China).
2.3. Syntheses of o-Vanillin-d-Phenylalanine (o-VDP),
o-Vanillin-l-Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL)
Schiff Bases
1.6519 g (0.01 mol) d-Phenylalanine and 0.5611 g (0.01 mol)
KOH were added to 50 mL ethanol solution in a 250 mL three-
neck flask and stirred for 3.0 h. And then, during reflux at
85 ◦C in oil bath, 50 mL o-Vanillin ethanol solution (contain-
ing 1.5215 g (0.02 mol) o-Vanillin) was dropwise added to above
d-Phenylalanine ethanol solution. After 3.0 h reflux, the mixed solu-
tion of d-Phenylalanine and o-Vanillin were concentrated to 10 mL
through reduced pressure distillation at 65 ◦C. After naturally cool-
ing to room temperature, the o-Vanillin-d-Phenylalanine (o-VDP)
(yellow microcrystals) Schiff Base appeared. The sample was fil-
trated, washed with ethanol three times and dried at 120 ◦C to
constant weight.
o-Vanillin-l-Tyrosine (o-VLT) (deep yellow microcrystals) and
o-Vanillin-l-Levodopa (o-VLL) (yellow brown microcrystals) Schiff
Bases were also synthesized according to the similar method above
mentioned. Their compositions were analyzed by elementalanal-
yser and the results were given in Table 1. The infrared spectra of
three o-VSB (o-VDP, o-VLT and o-VLL) were also determined by
using a Fourier transform infrared spectrophotometer. The corre-
sponding results were offered in Fig. 2.
2. Experimental
2.1. Reagents
Commercially prepared bovine serum albumin (BSA,
purity > 99.0%) was purchased from Beijing Abxing Biologi-
cal Technology Company and stored in refrigerator at 4.0 ◦C.
2-Hydroxy-3-methoxybenzaldehyde
(o-Vanillin,
analytical
reagent grade), 3-phenyl-d-alanine (l-Phenylalanine), 3-(4-
hydroxyphenyl)-l-alanine (l-Tyrosine, analytical reagent grade),
3-(3,4-dihydroxyphenyl)-l-alanine (l-Levodopa, analytical reagent
grade) were procured from Tianjing Tianhe Chemical Reagent Co.,
Ltd. o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine
(o-VLT) and o-Vanillin-l-Levodopa (o-VLL) Schiff Bases (o-VSB)
were synthesized through refluxing, distillation and filtration of
mixed solution of o-Vanillin with d-Phenylalanine, l-Tyrosine
and l-Levodopa, respectively. All other reagents were commer-
cial products of analytical grade and used as received. The Tris
(hydroxyl-methyl) aminomethane (Tris), HCl and NaCl were all of
analytical reagent grade, and double distilled water was used for
all solution preparation.
2.4. Measurement of binding parameters
For each of three synthesized o-VSB (o-VDP, o-VLT and o-
VLL), the binding parameters with BSA were measured by using
UV–vis and fluorescence spectroscopy. BSA and o-VSB stock solu-
tions were prepared in Tris–HCl–NaCl buffer solution (pH = 7.40
and[Tris–HCl] = [NaCl] = 0.05 mol/L)forkeepingthesolutionacidity
and ionic strength and their concentrations were 2.00 × 10−5 mol/L
2.2. Apparatus
The fluorescence measurements were performed with fluo-
rophotometer (Cary 300, Varian Company, USA) and the UV–vis
absorption spectra were recorded with UV–vis spectrophotometer
Table 1
Elementary analyses of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL.
Schiff bases
Molecular formula
C17 17NO4
Molecular weight
299.31
Melting point
194-204 ◦
C (%)
H (%)
N (%)
o-
VDP
o-
VLT
o-
VLL
Calcd.
Found
Calcd.
Found
Calcd.
Found
68.21
68.27
64.75
64.72
61.63
61.65
Calcd.
Found
Calcd.
Found
Calcd.
Found
5.72
5.77
5.43
5.46
5.17
5.14
Calcd.
Found
Calcd.
Found
Calcd.
Found
4.68
4.63
4.44
4.46
4.23
4.27
H
C
Nigger-brown above
250 ◦C
C17H17NO5
C17H17NO6
315.31
Carbonization above
331.31
250 ◦C

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J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
Fig. 2. Infrared spectra of o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL) (with potassium bromide (KBr) pellet at
25 ◦C).
Table 2
Quenching constants (KSV and Kq), binding constants, stable constants and binding site numbers calculated according to Stern–Volmer plots, Lineweaver–Burk plots and
Double logarithm plots of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions with o-VDP, o-VLT and o-VLL concentrations (from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at
0.50 × 10−5 mol/L intervals) ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
System
Stern–Volmer plot
R2
KSV (L/mol)
Kq (L/mol s)
BSA + o-VDP
BSA + o-VLT
BSA + o-VLL
F/F0 = 0.2471 [o-VSB] + 1
F/F0 = 0.1689 [o-VSB] + 1
F/F0 = 0.1332 [o-VSB] + 1
0.9986
0.9958
0.9991
2.47 × 104
1.69 × 104
1.33 × 104
2.47 × 1012
1.69 × 1012
1.33 × 1012
System
Lineweaver–Burk plot
R2
f
KLB (L/mol)
KD (mol/L)
BSA + o-VDP
BSA + o-VLT
BSA + o-VLL
1/[(F0 − F)/F0] = 4.5071/[o-VSB] + 0.7149
1/[(F0 − F)/F0] = 6.5776/[o-VSB] + 0.7279
1/[(F0 − F)/F0] = 7.9683/[o-VSB] + 0.7609
0.9982
0.9994
0.9994
0.7149
0.7279
0.7609
1.59 × 104
1.11 × 104
0.96 × 104
6.31 × 10−5
9.04 × 10−5
10.47 × 10−5
System
Double logarithm plot
R2
KA (L/mol)
n
ꢀG0 (kJ/mol)
BSA + o-VDP
BSA + o-VLT
BSA + o-VLL
log[(F0 − F)/F] = 1.0509 log[o-VSB] + 4.6344
log[(F0 − F)/F] = 1.0587 log[o-VSB] + 4.4945
log[(F0 − F)/F] = 1.0352 log[o-VSB] + 4.2905
0.9983
0.9985
0.9989
4.31 × 104
3.12 × 104
1.95 × 104
1.0509
1.0587
1.0352
−27.50
−26.67
−25.46
and 5.00 × 10−5 mol/L, respectively. In a 25.00 mL volumetric flask,
12.50 mL BSA stock solution and appropriate volume of o-VSB
stock solutions were added and diluted to the mark with the
same Tris–HCl–NaCl buffer solution. The final BSA concentration
was 1.00 × 10−5 mol/L, and the o-VSB concentrations were varied
from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L
intervals. The fluorescence spectra of BSA solutions along with
the increase of o-VSB concentrations were recorded in the wave-
length of 250–550 nm with excited wavelength at 280 nm and
5.0 nm/5.0 nm slit widths. All test solutions were incubated for
10 min before measurement. The curves of fluorescence quenching
spectra were got at 37.00 0.02 ◦C and given in Fig. 3. The maximal
intrinsic fluorescence intensities of BSA were recorded at 348 nm
for the calculation of quenching parameters. The corresponding
results were shown in Fig. 4 and Table 2. The binding distances
were determined according to Föster’s nonradiative energy trans-
fer theory (FRET). The spectral overlaps of fluorescence emission
of BSA solution and UV–vis absorption of o-VSB solutions were all
given in Fig. 5, and the corresponding parameters were offered in
Table 3.
2.5. Determination of synchronous fluorescence spectra
In order to confirm the binding sites of o-VSB (o-VDP, o-VLT
and o-VLL) to BSA molecules, the synchronous fluorescence spec-
Table 3
Energy transfer efficiency (E), critical binding distance (R), overlap integral
(J) and binding distance (r) calculated according to Foster’s non-radioactive
energy transfer theory ([BSA] = [o-VDP] = [o-VLT] = [o-VLL] = 1.00 × 10−5 mol/L,
[Tris–HCl] = [NaCl] = 50 mmol/L,
pH = 7.40,
Tsolu = 37.00 0.02 ◦
C
and
Vtotal = 25.00 mL).
System
E (%)
R (nm)
J (cm3 L/mol)
r (nm)
BSA + o-VDP
BSA + o-VLT
BSA + o-VLL
24.34
11.88
9.79
3.3083
3.3131
3.3407
7.12 × 10−15
7.18 × 10−15
7.55 × 10−15
3.9967
4.6266
4.8366

J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
1281
Fig. 3. Fluorescence spectra of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions with o-VDP, o-VLT and o-VLL concentrations (from 0.00 × 10−5 mol/L (o) to 2.50 × 10−5 mol/L
(e) at 0.50 × 10−5 mol/L intervals) ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
tra of BSA solutions along with the increase of o-VSB concentration
from 250 to 350 nm for ꢀꢁ = 15 nm and from 225 to 325 nm for
ꢀꢁ = 60 nm were determined. The corresponding results were all
given in Fig. 6. And the ratios of synchronous fluorescence quench-
ing were shown in Fig. 7.
3.2. Fluorescence spectra of BSA + o-Vanillin-d-Phenylalanine
(o-VDP), BSA + o-Vanillin-l-Tyrosine (o-VLT) and
BSA + o-Vanillin-l-Levodopa (o-VLL) solutions
In general, most of protein molecules in aqueous solution intrin-
sically fluoresce around 348 nm (with 278 nm excitation), which
is mainly attributed to the tryptophan (Trp), tyrosine (Tyr) and
phenylalanine (Phe) residues [48,49]. From Fig. 3 it can be seen that
the intrinsic fluorescence of bovine serum albumin (BSA) molecules
also appears around 348 nm. However, for all three courses along
with the addition of o-VSB (o-VDP, o-VLT and o-VLL) the intrinsic
fluorescences of (BSA) are gradually quenched compared with that
of pure BSA solution. Apparently, this is owing to the interaction of
o-VSB with BSA molecules in aqueous solution.
3. Results and discussion
3.1. The infrared spectra of o-Vanillin-d-Phenylalanine (o-VDP),
o-Vanillin-l-Tyrosine (o-VLT) and o-Vanillin-l-Levodopa (o-VLL)
Schiff Bases
The comparison of infrared spectra reveals the reaction between
o-Vanillin and d-Phenylalanine [46,47]. From Fig. 2 it can be seen
that the ꢂ(C O) of o-Vanillin at 1639 cm−1 and the ꢂas(NH3
)
+
3.3. Binding constant and binding site number
at 3065 cm−1 and ꢂs(NH3+) at 2546 cm−1 of d-Phenylalanine
disappears, while a new peak appears at 1618 cm−1. It indi-
cates that the C N bond of o-Vanillin-d-Phenylalanine (o-VDP)
Schiff Base was formed. Similarly, the ꢂas(NH3+) at 3066 cm−1
and ꢂs(NH3+) at 2554 cm−1 for l-Tyrosine and at 3067 cm−1 and
2551 cm−1 for l-Levodopa all disappear. And that at 1634 cm−1
and 1638 cm−1 new peak occur for o-Vanillin-l-Tyrosine (o-
VLT) and o-Vanillin-l-Levodopa (o-VLL) samples, respectively.
It also indicates the formation of o-Vanillin-l-Tyrosine (o-VLT)
and o-Vanillin-l-Levodopa (o-VLL) Schiff Bases. For all three
o-Vanillin Schiff Bases (o-VSB) there are broad ꢂ(OH) absorp-
tion bands around 3200 cm−1, revealing the existence of OH
bonds.
Being similar to many reported studies on the interaction of
small molecules with BSA [50–55], the interaction of o-VSB with
BSA molecules can also be investigated by measuring the change
of the intrinsic fluorescence along with the increase of o-VSB con-
centrations. Firstly, as a hypothetical dynamic quenching process,
the data of fluorescence intensities were analyzed by using the
Stern–Volmer equation (1) [50]:
F0
= 1 + KSV[o-VSB] = 1 + Kqꢃ0[o-VSB]
(1)
F
where the F0 and F are the fluorescence intensities of BSA solutions
at 348 nm, respectively, in the absence and presence of o-VSB with

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J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
Fig. 4. Stern–Volmer plot (a), Lineweaver–Burk plot (b) and Double logarithm plot (c) of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions with o-VDP, o-VLT and
o-VLL concentrations (from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L intervals) ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40,
Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
various concentrations. Kq is the apparent quenching constant of
bimolecular fluorescence, ꢃ0 is the life time of the fluorophore, KSV
is the Stern–Volmer fluorescence quenching constant and [o-VSB]
is the concentration of o-VSB (o-VDP, o-VLT and o-VLL).
The F0 and F are also the fluorescence intensities of BSA solu-
tions at 348 nm in the absence and presence of o-VSB (o-VDP,
o-VLT and o-VLL) with various concentrations. The
f is the
fraction of accessible fluorescence, and the KLB is the static
fluorescence quenching association constant. From the corre-
sponding plots in Fig. 4(b), the f and KLB could be obtained
(f = 0.7149 and KLB = 1.59 × 104 L/mol for BSA + o-VDP, f = 0.7279
and KLB = 1.11 × 104 L/mol for BSA + o-VLT and f = 0.7609 and
KLB = 0.96 × 104 L/mol for BSA + o-VLL). Correspondingly, the dis-
sociation constants (KD) are 6.31 × 10−5 mol/L for BSA + o-VDP,
9.04 × 10−5 mol/L for BSA + o-VLT and 10.47 × 10−5 mol/L for
BSA + o-VLL. In addition, from Table 2, it could be found that
all of these Lineweaver–Burk plots have a better linear relation-
ship (R2 = 0.9982 for BSA + o-VDP, R2 = 0.9994 for BSA + o-VLT and
R2 = 0.9994 for BSA + o-VLL) than the corresponding Stern–Volmer
plots. Thus, it could be confirmed again that the fluorescence
quenching mechanism of o-VSB (o-VDP, o-VLT and o-VLL) to
BSA is mainly a static quenching procedure indeed. Just like the
results calculated by Stern–Volmer equation, the KLB array order is
KLB(BSA + o-VDP) > KLB(BSA + o-VLT) > KLB(BSA + o-VLL). And that the KD array
Fig. 4(a) displays the Stern–Volmer plots of BSA + o-VSB solu-
tions. It can be found that three plots all exhibit a comparatively
good linear relationship (R2 = 0.9986 for BSA + o-VDP, R2 = 0.9958
for BSA + o-VLT and R2 = 0.9991 for BSA + o-VLL). The corresponding
plot expressions were shown in Table 2. According to Stern–Volmer
equation (1), for these three systems their Ksv could be obtained
(Ksv = 2.47 × 104 L/mol for BSA + o-VDP, Ksv = 1.69 × 104 L/mol for
BSA + o-VLT and Ksv = 1.33 × 104 L/mol for BSA + o-VLL). In gen-
eral, for the most of protein molecules, the ꢃ0 is known to be
approximately 10−8 s. Therefore, based on the Kq = Ksv/ꢃ0, the
Kq could be calculated (Kq = 2.47 × 1012 L/mol s for BSA + o-VDP,
Kq = 1.69 × 1012 L/mol s for BSA + o-VLT and Kq = 1.33 × 1012 L/mol s
for BSA + o-VLL). It can be seen that all of the above Kq values were
greater than the maximum value (2.00 × 1010 L/mol s) of the diffu-
sion controlled quenching process of biological macromolecules. It
provides the preliminary evidences that the dominating quench-
ing mechanism is not dynamic but static. The array order is
Kq(BSA + o-VDP) > Kq(BSA + o-VLT) > Kq(BSA + o-VLL), which indicates that the
interaction degree of o-VSB to BSA relates to the amino acid parts
in o-VSB Schiff Bases.
order is KD(BSA + o-VDP) < KD(BSA + o-VLT) < KD(BSA + o-VLL)
.
From Fig. 4(c), the equilibrium constants (KA) and the binding
site numbers (n) could be calculated by using the Double logarithm
equation (3):
ꢀ
ꢁ
For reconfirming the static fluorescence quenching mechanisms
of o-VSB (o-VDP, o-VLT and o-VLL) to BSA, the data of fluorescence
quenching are analyzed again according to the Lineweaver–Burk
(modified Stern–Volmer or double reciprocal) equation (2).
F0 − F
log
= log KA + n log[o-VSB]
(3)
F
Apparently, the KA and n could be measured, respectively, from
the intercept and slope obtained through plotting log[(F0 − F)/F]
against log[o-VSB]. It could be seen that the plots also
exhibited a good linear relationship (R2 = 0.9983 for BSA + o-
1
1
1
=
+
(2)
(F0 − F)/F0
fKLB[o-VSB]
f

J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
1283
Fig. 5. Spectral overlap of fluorescence (ꢁex = 278 nm) of BSA solution and absorption of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions ([BSA] = [o-VDP] = [o-VLT] = [o-
VLL] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
VDP, R2 = 0.9985 for BSA + o-VLT and R2 = 0.9989 for BSA + o-
VLL). Further, KA = 4.31 × 104 L/mol and n = 1.0509 for BSA + o-
VDP, KA = 3.12 × 104 L/mol and n = 1.0587 for BSA + o-VLT and
KA = 1.95 × 104 L/mol and n = 1.0352 for BSA + o-VLL were obtained.
Obviously, being similar to the KLB, the same array order is
KA(BSA + o-VDP) > KA(BSA + o-VLT) > KA(BSA + o-VLL). It illustrates that the
increase of hydroxyl group number on the benzene ring weakens
the interaction of o-VLT and o-VLL with BSA molecules. Maybe, it
is because that the hydroxyl group not only hinders the aromatic
ring stacking but also decreases the electrostatic bonding between
o-VLT and o-VLL with BSA molecules. Even though, the three KA
indicate that the bindings can be formed more easily between BSA
and o-VSB (o-VDP, o-VLT and o-VLL). The three n values are equal
to about 1, indicating that there is one class of binding site for these
three o-VSB to BSA molecule.
3.4. Binding distances between BSA with
o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine
(o-VLT) and o-Vanillin-l-Levodopa (o-VLL) Schiff Bases
In the preceding paragraph, the interaction of three o-VSB (o-
VDP, o-VLT and o-VLL) with BSA molecules could be speculated
through the regular fluorescence quenching of BSA solutions along
with the increase of o-VSB concentration. The binding distances
between BSA and o-VSB (o-VDP, o-VLT and o-VLL) can be deter-
mined according to Foster’s non-radioactive energy transfer theory
[51,52]. Based on this theory, the efficiency (E) of energy transfer
between donor (BSA) and acceptor (o-VDP, o-VLT and o-VLL) can
be calculated by Eq. (5):
1
E =
(5)
1 + (r/R0)6
Utilizing KA, the free energy change (ꢀG0) can be calculated
from the relationship (4):
where r is the binding distance between donor and acceptor, and
R0 is the critical binding distance when the efficiency (E) of energy
transfer is 50%, which can be calculated by Eq. (6):
ꢀG0 = −RT ln KA
(4)
R06 = 8.8 × 10−25(K2n−4ϕDJ)
(6)
Here, R = 8.314 J/mol K and T = 310 K were fixed, and the ꢀG0 are
−27.50 kJ/mol for BSA + o-VDP, −26.67 kJ/mol for BSA + o-VLT and
−25.46 kJ/mol for BSA + o-VLL could calculated. The negative sign
for ꢀG0 indicates the binding spontaneity of three o-VSB (o-VDP,
o-VLT and o-VLL) to BSA molecules. Obviously, the same array order
where the K2 is the spatial orientation factor of the dipole, n is
the refractive index of medium, ϕD is the quantum yield of the
donor in the absence of acceptor and J is the overlap integral of the
emission spectrum of the donor and the absorption spectrum of the
acceptor. In the present case, K2, n and ϕD are 2/3, 1.336 and 0.15,
respectively, for BSA [53]. And then, the J can be calculated by Eq.
is found to be ꢀG0(BSA + o-VDP) < ꢀG0(BSA + o-VLT) < ꢀG0(BSA + o-VLL)
.
It demonstrates that the o-VDP binds more easily with BSA
molecules.

Fig. 6. Synchronous fluorescence spectra of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions with o-VDP, o-VLT and o-VLL concentrations (from 0.00 × 10−5 mol/L (o) to 2.50 × 10−5 mol/L (e) at 0.50 × 10−5 mol/L intervals)
([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).

J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
1285
Fig. 7. Ratios of synchronous fluorescence quenching (RSFQ
) of BSA + o-VDP, BSA + o-VLT and BSA + o-VLL solutions with o-VDP, o-VLT and o-VLL concentrations
(from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L intervals) ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and
Vtotal = 25.00 mL).
(7).
(o-VDP, o-VLT and o-VLL) to BSA molecules. The RSFQ were calcu-
lated by using RSFQ = 1 − F/F0 equation, in which F and F0 are the
synchronous fluorescence intensities of BSA in the presence and
absence of o-VSB (o-VDP, o-VLT and o-VLL), respectively.
˙F(ꢁ)ε(ꢁ)ꢁ4ꢀꢁ
˙F(ꢁ)ꢀꢁ
J =
(7)
where the ꢁ is the common wavelength of corresponding fluores-
cence spectrum of BSA and absorption spectrum of o-VSB (o-VDP,
o-VLT and o-VLL). The F(ꢁ) and ε(ꢁ) are the fluorescence intensity
of BSA solution and the absorbance of o-VSB (o-VDP, o-VLT and
o-VLL) solution, respectively. Fig. 5 shows the spectral overlap of
fluorescence emission of BSA solution and UV–vis absorption of o-
VSB (o-VDP, o-VLT and o-VLL) solutions in the wavelength range of
260–500 nm, respectively. The efficiency (E) of energy transfer can
be determined by Eq. (8):
From Fig. 6 it can be seen that for three courses the fluores-
cence intensities for ꢀꢁ = 15 nm (upper) and ꢀꢁ = 60 nm (nether)
are both quenched more and more seriously along with increas-
ing o-VSB (o-VDP, o-VLT and o-VLL) concentration. Fig. 7 markedly
reveals that, for both ꢀꢁ = 15 nm and ꢀꢁ = 60 nm, the array order
of RSFQ is BSA + o-VDP > BSA + o-VLT > BSA + o-VLL. It proves again
that the existence of hydroxyl group somewhat weaken the inter-
action of o-VLT and o-VLL with BSA molecules. In addition, for
BSA + o-VDP at any o-VDP concentration, the RSFQ for ꢀꢁ = 15 nm
are slightly bigger than corresponding ones for ꢀꢁ = 60 nm. By con-
traries, for BSA + o-VLT and BSA + o-VLL the RSFQ for ꢀꢁ = 15 nm
are slightly smaller than the corresponding ones for ꢀꢁ = 60 nm.
It indicates that the existence of hydroxyl group changes the inter-
action degree and mode of o-VLT and o-VLL with BSA molecules.
That is, these three o-VSB Schiff Bases (o-VDP, o-VLT and o-
VLL) affect the microenvironment of Tyr and Trp residues upon
binding to BSA molecules with different accessibility [57]. To
sum up, the o-VDP may be more accessibility to Tyr residues,
whereas the o-VLT and o-VLL do more accessibility to the Trp
residues. It illuminates that these three o-VSB Schiff Bases can
bind to BSA molecules with different opportunities to Tyr and Trp
residues.
F
E = 1 −
(8)
F0
where F0 and F are the fluorescence intensities of BSA solutions
with 1.0 × 10−5 mol/L at 345 nm in the absence and presence of o-
VSB (o-VDP, o-VLT and o-VLL) with 1.0 × 10−5 mol/L, respectively.
According to the above Eqs. (4)–(8), E, R0 and J could all be calcu-
lated and the corresponding results were given in Table 3. At the
same time, the binding distance (r) between BSA and o-VSB (o-VDP,
o-VLT and o-VLL) are obtained and the corresponding results are
3.9967 nm for BSA-o-VDP, 4.6266 nm for BSA-o-VLT and 4.8366 nm
for BSA-o-VLL, respectively. Being similar to the results of fluo-
rescence quenching, the array order of the binding distances (r)
is also r(BSA − o-VDP) < r(BSA − o-VLT) < r(BSA − o-VLL). Apparently, they are
all less than 7.0 nm, which indicates that the energy transfer from
BSA to o-VSB (o-VDP, o-VLT and o-VLL) occurs with high possi-
bility [54]. It also suggested that the bindings of these three o-VSB
Schiff Bases to BSA molecules were formed through energy transfer,
which quenched the fluorescence of BSA molecules.
4. Conclusions
The interaction of three synthesized o-Vanillin Schiff Bases
(o-VSB: o-Vanillin-d-Phenylalanine (o-VDP), o-Vanillin-l-Tyrosine
(o-VLT) and o-Vanillin-l-Levodopa (o-VLL)) with Bovine serum
albumin (BSA) molecules in aqueous solution were investigated by
fluorescence spectroscopy. The experimental results showed that
these o-VSB Schiff Bases can obviously bind to BSA molecules, and
then quench their intrinsic fluorescence efficiently. According to
the fluorescence quenching calculation, the bimolecular quenching
constant (Kq), apparent quenching constant (Ksv), effective bind-
ing constant (KA) and corresponding dissociation constant (KD),
binding site number (n) and binding distance (r) were obtained.
In comparison, the interaction strength of these three o-VSB Schiff
Bases with BSA molecules decreases as the array order o-VDP, o-VLT
and o-VLL. The synchronous fluorescence spectroscopy indicates
that the o-VDP is more accessibility to tryptophan (Trp) residues of
BSA molecules than to tyrosine (Tyr) residues, but the o-VLT and
3.5. Synchronous fluorescence spectra of
BSA + o-Vanillin-d-Phenylalanine (o-VDP),
BSA + o-Vanillin-l-Tyrosine (o-VLT) and
BSA + o-Vanillin-l-Levodopa (o-VLL) solutions
Synchronous fluorescence spectra can provide much valuable
information about the microenvironment around fluorogens in
biomolecules [55]. In general, for protein molecules, when the
ꢀꢁ = 15 nm is fixed, the spectrum characteristic of tyrosine (Tyr)
residues could be observed, and when ꢀꢁ = 60 nm is done, the spec-
trum characteristic of tryptophan (Trp) residues could be obtained
[53,54,56]. In this work, the ratios of synchronous fluorescence
quenching (RSFQ) were used to determine the binding sites of o-VSB

1286
J. Gao et al. / Spectrochimica Acta Part A 78 (2011) 1278–1286
o-VLL are more accessibility to Tyr residues of BSA molecules than
to Trp residues.
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